Sexual transmission of HIV occurs when cell-free or cell-associated virus infects cells primarily via the CCR5 coreceptor, expressed on macrophages, dendritic cells and activated T lymphocytes. A microbicide that could be used vaginally or anally to prevent sexual transmission would make a substantial contribution to controlling the spread of HIV. This innovative grant will begin to explore the hypothesis that RNA interference (RNAi) can form the basis of an effective anti-HIV microbicide. RNAi is an ancient, evolutionarily conserved, host defense against viruses and transposable elements, which uses small doublestranded RNAs, called small interfering RNAs (siRNA), to silence gene expression with exquisite specificity by targeted degradation of homologous mRNAs. There has been a lot of excitement about the therapeutic potential of RNAi to treat viral infection. A major obstacle is how to deliver siRNAs into cells. Our preliminary results suggest that duplex siRNAs can be delivered to macrophages and activated T cells without transfection and lead to prolonged gene silencing, which can inhibit de novo infection as well as viral replication in already infected cells. This suggests that duplex siRNAs might serve as the active component in a microbicide. Many steps are needed to determine whether an siRNA-based microbicide is possible. These include in vivo delivery of siRNAs to dendritic cells, macrophages, and (if possible) lymphocytes in the genital mucosa of small animals, and demonstration that delivered siRNAs effectively inhibit HIV production. Delivery methods have to be safe and compatible with a formulation suitable for vaginal and/or anal delivery that does not induce inflammation at the mucosa. Early proof-of-principle studies eventually need to be complemented by formal pharmacokinetics, toxicity and efficacy studies in small animals and primates. This grant will focus on testing methods to deliver duplex siRNA to macrophages and dendritic cells in vitro and in vivo in mice and in collaboration with A. Blauvelt of the NIH in a human ex vivo skin blister model for the female genital epithelium, siRNA delivery in vitro using PBMCs from the pig-tailed macaque Macaca nemestrina will also be studied, in preparation for later delivery, toxicity and challenge studies in this macaque model. Delivery methods will be compared for their ability to induce a silenced state resistant to HIV/SHIV infection in vitro. The specific aims are to: 1. optimize strategies to deliver duplex siRNAs that silence CCR5 to mouse macrophages, dendritic cells and T cells in vitro and in the female mouse genital mucosa; 2. test siRNA delivery strategies in the human skin blister model; 3. test siRNAs and siRNA delivery strategies for silencing SIV gag and CCR5 in PBMCs from pig-tailed macaques.